Potassium ion (K.sup.+) channels comprise a diverse family of membrane proteins that regulate action potentials, cardiac pacemaking, and neurotransmitter release in excitable tissues. In non-excitable tissues, these channels play important roles in hormone secretion, cell proliferation, cell volume regulation, and lymphocyte differentiation. These diverse and significant potassium channel functions have stimulated researchers to investigate potassium channels at the molecular level.
As a result of this research, it was found that a functional potassium channel has four identical and/or homologous polypeptides, generally referred to as .alpha.-subunits, that together form a central conduction pore, or channel, for potassium ions. As there are many types of .alpha.-subunits, the aforesaid functional diversity of the potassium channels arises primarily from the particular combination of .alpha.-subunits present in a particular potassium channel.
Another significant influence on the function of potassium channels is the regulatory interactions of the .alpha.-subunits with another type of polypeptide, a hydrophilic polypeptide commonly referred to as .beta.-subunit. Thus, a combination of the particular .alpha.-subunits in a particular potassium ion channel, and the interaction of that channel with a .beta.-subunit, allows individual cells to acquire their own characteristic potassium current properties.
One well-known type of potassium ion channel is a Shaker-like channel. These channels are distinguished from other types of potassium channels by the presence of an .alpha.-subunit having a hydrophobic core region composed of six transmembrane spanning domains (S1 to S6) flanked by cytoplasmic amino (NH.sub.2)-- and carboxyl(COOH)-terminal domains. The .alpha.-subunits present in the Shaker-like channels are encoded by one or more .alpha.-subunit genes. These genes have been divided into at least five subfamilies: Kv1 (Shaker), Kv2(Shab), Kv3 (Shaw), Kv4 (Shal), and Kv5.
One limitation on the structure of Shaker-like potassium ion channels is that .alpha.-subunits can only form channels with other .alpha.-subunits that were expressed using a gene from the same subfamily. In conducting systematic binding studies to investigate this phenomenon, it was found that there exists a highly conserved region within the NH.sub.2 -terminal domain of each Shaker-like .alpha.-subunit that mediates this subfamily-specific association. This conserved region is commonly referred to as the NH.sub.2 -terminal A and B box (NAB) (Drewe et al., J. Neurosci., 12, 538-548 (1992), see also Aldrich Current Biology, 4, 839-840 (1994). Thus, due to their homology, the NAB's were determined to be the region of the .alpha.-subunits that enabled certain other .alpha.-subunits to associate with one another and thereby provide a basis for the number of functionally-diverse potassium channels.
While the interaction between the .alpha.-subunits with one another has been studied, the information regarding the impact of the differing .alpha.-subunit combinations on potassium channel properties remains limited. This lack of information is even greater with respect to the regulation of potassium channels by the .beta.-subunits. The .beta.-subunits were originally identified in membrane preparations from bovine brain as soluble proteins, and this discovery has led to the isolation of genes that encode two such .beta.-subunits, Kv.beta.1 and Kv.beta.2(Rettig et al., Nature, 369, 289-294 (1994); Scott et al., Proc. Natl. Acad. Sci. USA, 91, 1637-1641 (1994)). Although very little is known about the function of .beta.-subunits, recent studies have revealed that a particular .beta.-subunit (that encoded by Kv.beta.1), when introduced into Xenopus oocytes, functions to help close the ion pathway in one type of potassium ion channel in a relatively rapid manner. This accelerated pathway closure is referred to as N-type inactivation. However, the method by which this .beta.-subunit accomplishes this inactivation is not well understood. Moreover, and more generally, other than as discussed previously, little is known about potassium ion channel regulation at the molecular level, including the existence and identification of component interactions that may play a role in such regulation.
Due to the importance of potassium ion channels in regulating clinically relevant characteristics of human health, such as heart rate, research has focused on identifying compounds which affect the function of potassium ion channels. By way of example, some classes of disorders that may be affected by effective manipulation of Shaker-like potassium ion channels include neurological disorders, tumor driven diseases, metabolic diseases, cardiac diseases, and autoimmune diseases. Examples of disease states and conditions from these and other classes, as well as affected normal body functions, encompass: hypoglycemia, anoxia/hypoxia, renal disease, osteoporosis, hyperkalemia, hypokalemia, hypertension, Addison's disease, abnormal apoptosis, induced apoptosis, clotting, modulation of acetylcholine function, and modulation of monoaminesepilepsy, allergic encephalomyelitis, multiple sclerosis (any demylelinating disease), acute traverse myelitis, neurofibromatosis, cardioplegia, cardiomyopathy, ischemia, ischemia reperfusion, cerebral ischemia, sickle cell anemia, cardiac arrythmias, peripheral monocuropathy, polynucuropathy, Gullain-Barre' Syndrome, peroneal muscular dystrophy, neuropathies, Parkinson's disease, palsies, cerebral palsy, progressive supranuclear palsy, pseudobubar palsy, Huntington's disease, dystonia, dyskinesias, chorea, althetosis, choreothetosis, tics, memory degeneration, taste perception, smooth muscle function, skeletal muscle function, sleep disorders, modulation of neurotransmitters, acute disseminated encephalomyelitis, optic neuromyelitis, muscular dystrophy, myasthenia gravis, multiple sclerosis, and cerebral vasospasm, hypertension, angina pectoris, asthma, congestive heart failure, ischemia related disorders, cardiac dysrhythmias, diabetes, carcinomas, neurocarcinomas, autoimmune-hypertrophy, neuromyotonia (Isaac's Syndrome) muscular disorders associated with drug abuse, and treatment for poisoning. The search for finding such compounds involves tedious, difficult, and/or insensitive biochemical methods that measure either the ability of a compound to bind to an ion channel (e.g., chromatography), or the ability of a compound to alter a physiological response (e.g., voltage clamp recording).
A system that has been used for measuring the interactions of two proteins is the yeast two-hybrid system. In this system, cDNA libraries or other suitable sources of protein coding sequences are expressed individually, in individual cells as fusion proteins with either a DNA-binding domain or a transactivation domain. There are, however, difficulties in using this system with respect to a particular protein of interest. For example, a particular protein to be studied may have a binding region for other proteins which is comprised of non-contiguous polypeptide sequences. In this case, it would be very difficult to identify an appropriate polypeptide to measure the protein-protein interactions. Additionally, one may find that the region of polypeptide to be used in the two hybrid system does not retain a structure similar to that found in the whole protein. Further, the polypeptide selected can be toxic to the host yeast, or can have intrinsic transcriptional activation potential. In each of these cases, the implementation of the yeast two-hybrid system would be difficult. Further, there exists no reliable indicator one may use to predict whether one could expect to encounter the aforementioned problems with respect to a particular polypeptide.
In view of the foregoing problems, there exists a need for compounds and related methods of artificially modulating potassium channels, and for an economical and efficient means of identifying such compounds. The present invention provides such compounds and related methods of modulating Shaker-like potassium ion channels, as well as an assay for identifying such compounds. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.